Therapeutic compositions (II)

ABSTRACT

Compositions comprising ketone bodies and/or their metabolic precursors are provided that are suitable for administration to humans and animals and which have the properties of, inter alia, (i) increasing cardiac efficiency, particularly efficiency in use of glucose, (ii) for providing energy source, particularly in diabetes and insulin resistant states and (iii) treating disorders caused by damage to brain cells, particularly by retarding or preventing brain damage in memory associated brain areas such as found in Alzheimer&#39;s and similar conditions.  
     These compositions may be taken as nutritional aids, for example for athletes, or for the treatment of medical conditions, particularly those associated with poor cardiac efficiency, insulin resistance and neuronal damage. The invention further provides methods of treatment and novel esters and polymers for inclusion in the compositions of the invention.

[0001] The present invention relates to compositions suitable foradministration to humans and animals which have the properties ofincreasing levels of (R)-3-hydroxybutyrate ((R)-3-hydroxybutyric acid orD-β-hydroxybutyrate) when so administered; particularly whenadministered orally, topically, subcutaneously or parenterally, but mostadvantageously orally.

[0002] Administration of (R)-3-hydroxybutyric acid has a number ofbeneficial actions on the human and animal body. These include interalia, increasing cardiac efficiency, e.g. in heart failure, provision ofan alternative energy source to glucose, e.g. in diabetes and insulinresistant states, and treating disorders caused by damage to neuronalcells, e.g. CNS cells, particularly by retarding or preventing braindamage such as found in Alzheimer's and Parkinsonism and similardiseases and conditions.

[0003] Sodium hydroxybutyrate has been shown to increase cerebralcirculation and regional vasomotor reflexes by up to 40% (Biull. Eksp.Biol. Med Vol 88 11, pp 555-557). EP 0780123 A1 further teaches use ofacetoacetate, β-hydroxybutyrate, monohydric, dihydric or trihydricalcohol esters of these or linear oligomers of 2 to 10 repeats ofβ-hydroxybutyrate for suppressing cerebral edema, protecting cerebralfunction, rectifying cerebral energy metabolism and reducing the extentof cerebral infarction.

[0004] Intravenous infusion of sodium salts of (R)-3-hydroxybutyrate hasbeen performed on normal human subjects and patients for a number ofconditions, e.g. those undergoing treatment for severe sepsis in anintensive care unit and is found to be non-toxic and capable ofdecreasing glucose free fatty acids and glycerol concentration, butineffective in decreasing leucine oxidation.

[0005] The present inventor has further determined that compounds andcompositions that raise blood levels of (R)-3-hydroxybutyric acid and/oracetoacetate also have utility in reducing free radicals in vivo, andthus have a place in treatment of free radical associated diseases.

[0006] (R)-3-hydroxybutyrate and acetoacetate, commonly referred to asketone bodies, provide a normal physiological alternative to the usualenergy producing substrates, glucose and fatty acids. During prolongedfasting in man fatty acids are converted by liver to(R)-3-hydroxybutyric acid and acetoacetate which can be utilized by mostmajor tissues of the body except liver. Under these conditions, totalblood ketone bodies are elevated to about 7 mM. When these are modestlyelevated in the blood, extrahepatic tissues such as brain, heart andskeletal muscle utilize these ketone bodies within the mitochondria toprovide reducing power in the form of NADH which is the primarysubstrate of the electron transport system and generator of the energyrequired for the synthesis of ATP. In turn, generation of mitochondrialNADH by ketones, lowers the ratio of free mitochondrial [NAD⁺]/[NADH]ratio and the cytosolic [NADP⁺]/[NADPH] ratio to which the mitochondrial[NAD⁺]/[NADH] is linked. While the catabolism of ketones reducesmitochondrial [NAD⁺]/[NADH] it oxidizes the ratio of mitochondrial[ubiquinone]/[ubiquinol], [Q]/[QH₂]. The semiquinone form of ubiquinolis the major source of the generation by mitochondria of superoxide, O₂⁻. By decreasing the amount of the reduced form QH₂, and itssemiquinone, one can decrease the generation of free radicals bymitochondria while at the same time increasing the scavangers of freeradicals linked to the NADP system, such as glutathione.

[0007] The inventor has thus determined that free radical damageresulting from excess reduced Q or inhibition of NADH dehydrogenase,such as occurs in MPP induced toxicity, can be reduced by administrationof agents which elevate ketone body levels in vivo.

[0008] A number of disease processes involve damage by free radicalsamong which are the neurological diseases: Parkinson's disease,amyotrophic lateral sclerosis, Alzheimer's disease and cerebralischemia. In addition excessive free radical damage has been implicatedas playing a role in coronary reperfusion, diabetic angiopathy,inflammatory bowel disease and pancreatitis.

[0009] The inventor's copending WO 98/41201 discloses the administrationof linear esters of (R)-3-hydroxybutyric acid and/or acetoacetate inproducing elevated levels of the free compounds in vivo. Oraladministration of 4 mM solutions of the oligomertetra-(R)-3-hydroxybutyrate or its acetoacetyl ester was shown to raiseblood levels of ketone bodies such that (R)-3-hydroxybutyrate levelscould be measured to have increased by 1 to 2 mM for periods in excessof 2 hours.

[0010] The inventor has now determined that unexpected advantages areprovided when the (R)-3-hydroxybutyric acid component of suchcomposition is administered as a cyclic oligomer. These advantages mayinclude, inter alia, (a) increased efficiency in raising blood(R)-3-hydroxybutyric acid levels such that levels may be increased bymore than 2 mM, including attainment of near fasting levels and beyond,(b) maintenance of elevated levels for periods of several hours, (c)ability to be administered without counterion, such as sodium ormethylglucamine, where it is desirable not to increase a patient's saltload or where significant dosing is envisaged and (d) relative ease ofmanufacture of pure compound from polymeric starting materials availablethrough bioculture.

[0011] The present application particularly addresses the problem ofneurodegenerative diseases, particularly disease where neurons aresubject to neurotoxic effects of pathogenic agents such as proteinplaques and oxidative damage and further provides compositions for usein treating these and the aforesaid disorders.

[0012] In preferred embodiments the present invention provides elevationof blood ketones necessary to correct the defects described above andcan be accomplished by parenteral or enteral administration.Particularly it does not require the administration of potentially toxicpharmacological agents. The present invention's improved efficacy inraising levels, particularly blood levels, of ketone bodies providestherapeutic effects of the classical ketogenic diet, which is not itselffound to be toxic in children, with none of the side effects that renderthat unused adults. Furthermore, the inventor has determined that withthe correction of the aforesaid metabolic and toxic defects, cytokineresponses and the increase in apoptotic peptides in degenerating cellswill decrease due to the increase in neuronal cell energy status and theincreased trophic stimulation resulting from increased neurotransmitter,e.g. acetyl choline, synthesis.

[0013] The treatment that the present inventor provides goes beyondketone body effects on circulation, as it provides treatment for cellsthat are unable to function due to neuro-degeneration and/or metabolicdefects, particularly in metabolism of glucose, e.g. caused byneurotoxic agents such as peptides, proteins, free radical damage andeffect of genetic abnormality. The treatment involves action of ketonebodies on the cells themselves and not the flow of blood to them.

[0014] Thus in a first aspect of the present invention there is provideda cyclic ester of (R)-3-hydroxybutyrate of formula (I)

[0015] where n is an integer of 1 or more

[0016] or a complex thereof with one or more cations or a salt thereoffor use in therapy or nutrition.

[0017] For oral delivery free cyclic oligomer may be preferred. Wherecations are present in a complex preferred cations are sodium,potassium, magnesium and calcium, and are balanced by physiologicallyacceptable counter-anion providing a salt complex.

[0018] Examples of typical physiologically acceptable salts will beselected from sodium, potassium, magnesium, L-Lysine and L-arginine ore.g. more complex salts such as those of methyl glucamine salts.

[0019] Preferably n is an integer from 1 to 200, more preferably from 1to 20, most preferably from 1 to 10 and particularly conveniently is 1,i.e. (R, R, R)-4,8,12-trimethyl-1,5,9-trioxadodeca-2,6,10-trione, 2,3,4or 5.

[0020] The cyclic esters of the invention are preferably used in thetreatment of disease states mediated by free radicals, toxic agents suchas peptides and proteins, genetic defects detrimental to nerve cellmetabolism, insulin resistance or other glucose metabolism defects ordefect inducing states, ishemia, head trauma and/or for increasing cellefficiency, e.g. cardiac cell efficiency e.g. in heart failure.

[0021] A second aspect of the invention provides methods of treatingcells that are subject to malfunction due to action of free radicals,toxic agents such as peptides and proteins, genetic defects detrimentalto cell metabolism, insulin resistance or other glucose metabolismdefects or defect inducing states, ischemia, head trauma and/or forincreasing cell efficiency characterised in that it comprisesadministration of a cyclic oligomer of formula (I). This may includetreatment of such disease states in humans and/or animals.

[0022] This aspect includes such use as a neuronal stimulant e.g.capable of stimulating axonal and/or dendritic growth in nerve cells,e.g. in hippocampal or substantia nigral cells, in vivo or in vitro,particularly in conditions where neurodegeneration has serious clinicalconsequences, through its elevating effect on blood and plasma(R)-3-hydroxybutyrate and acetoacetate levels.

[0023] A third aspect of the invention provides a method of enteral orparenteral nutrition, preferably oral route nutrition, comprisingadministration of a cyclic oligomer of formula (I).

[0024] A fourth aspect of the invention provides the use of a cyclicester formula I for the manufacture of a medicament for the treatment ofdisease states mediated by free radicals, toxic agents such as peptidesand proteins, genetic defects detrimental to cell metabolism, insulinresistance or other glucose metabolism defects or defect inducingstates, ischemia, head trauma and/or for increasing cell efficiency.

[0025] A fifth aspect of the invention provides compositioncharacterised in that it comprises a cyclic oligomer of formula (I) inphysiologically acceptable form e.g. with a physiologically acceptablecarrier.

[0026] Particularly the composition is suitable for parenteral orenteral administration, particularly for oral administration. Where thecomposition is for parenteral use it is sterile and pyrogen free. Fororal use the composition may include a foodstuff base and may be in theform of an emulsion or mere admixture with solid food.

[0027] Particularly the cyclic oligomer(s) comprise an effective amountof the total composition, e.g. at least 2% or more, e.g. at least 5%, ofthe composition by weight, more preferably 20% or more and mostpreferably 50% to 100%. The composition may be adapted for oral,parenteral or any other conventional form of administration.

[0028] In preferred forms of all of the aspects of the invention thecompound of formula (I) is administered together with a physiologicalratio of acetoacetate or a metabolic precursor of acetoacetate. The termmetabolic precursor thereof particularly relates to compounds thatincorporate acetoacetyl moieties such as acetoacetyl-1,3-butandiol,preferably acetoacetyl-(R)-1,3-butandiol,acetoacetyl-(R)-3-hydroxybutyrate, and acetoacetylglycerol. Esters ofany such compounds with monohydric, dihydric or trihydric or higher,e.g. glucosyl, alcohols are also envisaged.

[0029] In diabetic patients this use of the cyclic oligomers allowsmaintenance of low blood sugar levels without fear of hypoglycemiccomplications. In normal non-diabetic subjects the fasting blood sugaris 80 to 90 mg % (4.4-5 mM) rising to 130 mg % (7.2 mM) after a meal. Indiabetics ‘tight control’ of diabetes has long been recommended as amethod for retardation of vascular complications but, in practice,physicians have found it difficult to keep blood sugars tightlycontrolled below 150 mg % (8.3 mM) after eating because of hypoglycaemicepisodes. Hypoglycaemic coma occurs regularly in normal subjects whoseblood sugar drops to 2 mM. As discussed earlier, (62, 63) in thepresence of 5 mM blood ketones there are no neurological symptoms whenblood sugars fall to below 1 mM.

[0030] The present inventor has determined that supplementing type IIdiabetics with cyclic oligomers of the invention will allow bettercontrol of blood sugar, thus preventing the vascular changes in eye andkidney which occur now after 20 years of diabetes and which are themajor cause of morbidity and mortality in diabetics.

[0031] Where the therapy is aimed at seizure related disorders, such asrefractory epilepsy as is treated by the ketogenic diet, therapy isimproved by use of cyclic oligomers, due to the reduction or eliminationof both high lipid and carbohydrate content. Such patients include thosewith genetic defects in the brain glucose transporter system, inglycolysis or in PDH itself such as in Leigh's syndrome, endotoxic shockor high stress states.

[0032] Particular disorders treatable with these medicaments areapplicable to all conditions involving PDH blockage, including thoseconditions occuring after head trauma, or involving reduction orelimination of acetyl CoA supply to the mitochondrion such as insulincoma and hypoglycaemia, defects in the glucose transporter in the brain,or elsewhere (80), or in glycolytic enzyme steps.

[0033] Where the medicament or nutraceutical comprises acetoacetate itis preferably not stored for a prolonged period or exposed totemperatures in excess of 40° C. Acetoacetate is unstable on heating anddecomposes violently at 100° C. into acetone and CO₂. In suchcircumstances it is preferred that acetoacetate is generated by thecomposition on contact with the bodies metabolic processes. Preferablythe composition comprises an ester precursor of actetoacetate.

[0034] A sixth aspect of the invention provides a method of treating ahuman or animal neuronal cell, e.g. brain cells, subject to cell damagerelated disorder, particularly those which lead to cell death, asreferred to for the second to fourth aspects, particularly aneurodegenerative disorder e.g. such as those related to neurotoxicconditions such as presence of amyloid protein, e.g. a memory ormovement associated disorder such as Alzheimer's or Parkinson'sdiseases, or epileptic seizures, comprising administering to that personat least one of the materials for use in the first to fifth aspects ofthe invention.

[0035] The inventor has further determined that ketone bodies, providedby administration of the cyclic oligomers of (R)-3-hydroxybutyric acidin amounts sufficient to raise total blood ketone body concentration toelevated levels result in more than simple maintenance of cell viabilitybut actually improve cell function and growth beyond that of normal,i.e. control levels in a manner unrelated to blood flow or nutrition. Inthis respect the invention further provides use of the cyclic oligomersas agents capable of producing neuronal stimulation, i.e. nerve growthfactor like activity, increase of metabolic rate and increase of extentof functional features such as axons and dendrites. This aspect of thepresent invention offers a mechanism for improvement of neuronalfunction as well as mere retardation of degredation.

[0036] The recent work of Hoshi and collaborators (77, 78) stronglysuggests that a part of the amyloid protein whose accumulation is thehallmark of Alzheimer's disease, Aβ₁₋₄₂, acts to stimulate mitochondrialhistidine protein kinase which phosphorylates and inactivates thepyruvate dehydrogenase multienzyme complex. The PDH complex is amitochondrial enzyme responsible for the generation of acetyl CoA andNADH from the pyruvate produced by glycolysis within the cytoplasm. Themitochondrial acetyl CoA formed condenses with oxaloacetate to start theKrebs TCA cycle completely combusting pyruvate to CO₂ while providingthe mitochondria with the reducing power which becomes the substrate forthe electron transport system through which the energy required formitochondrial ATP synthesis is generated

[0037] Ketone body utilization in brain is limited by the transport,with lesser utilization occurring in the basal ganglion at blood levelsbelow 1 mM (76). However, at levels of 7.5 mM achieved in normal man byprolonged fasting, the rate of ketone body entry into brain issufficient to take over the majority of cerebral energy needs and toprevent hypoglycemic symptoms, even in the face of blood sugar levelswhich would normally cause convulsions or coma (63) .

[0038] In the copending application WO 98/41201, ‘Therapeuticcompositions’, it is the inventor's hypothesis that in Alzheimer'sdisease, where there is a block at PDH which prevents the normal energyproduction from glucose, if one can provide elevated, e.g. normalfasting levels of ketones, one can bypass the PDH blockade present inthese patients thereby preventing cell death due to energy depletion orlack of cholinergic stimulation and thus slow the progression of thememory loss and dementia. Furthermore, utilising the nervegrowth/stimulatory effects of the ketone bodies, particularly(R)-3-hydroxybutyrate or a physiological ratio of this withacetoacetate, cells that are still viable can be caused to improvebeyond the state to which they have degenerated and accordingly someimprovement of function will be seen in patients.

[0039] In fed animals and in man the liver content, which is essentiallythat of blood, of acetoacetate is very low, e.g. 0.09 mM and(R)-3-hydroxybutyrate is 0.123 mM but rises after a 48 hour fast to e.g.0.65 mM acetoacetate and 1.8 mM (R)-3-hydroxybutyrate (84). The ketonebodies rise in starvation because the fall in insulin decreases there-esterification of fatty acids to triglyceride in adipose tissuecausing the release of free fatty acids into the blood stream. Thereleased free fatty acids can then be taken up and used as a source ofenergy by muscle, heart, kidney and liver in the process of β-oxidation.Liver, however, has the capacity to convert the free fatty acids to ametabolic fuel, ketones, for use by extra-hepatic organs, including thebrain, as an alternative to glucose during periods of fasting. Thehepatic synthesis of ketone bodies occurs from mitochondrial acetyl CoAgenerated during the β-oxidation of fatty acids by liver.

[0040] The ketone bodies enter extra-hepatic tissues on the samecarrier, where other monocarboxylates can act as competitive inhibitors.Unphysiological isomers such as D-lactate or (S)-3-hydroxybutyrate canalso act as competitive inhibitors to ketone body transport. Sinceketone body transport across the blood brain barrier is a limitingfactor to ketone body utilization in brain (76) every effort should bemade to keep the blood concentration of these unphysiologicalenantiomers at low levels during ketogenic therapy. When blood ketonebody concentrations are elevated to levels found in starvation, heart,muscle, kidney and brain utilize ketone bodies as the preferred energysubstrate.

[0041] The present inventor has thus determined that the mitochondrialacetyl CoA derived from ketone bodies as produced using the cyclicoligomers taught by the present invention can thus replace the acetylCoA deficiency which occurs during inhibition of PDH multienzyme complexin tissues dependent upon the metabolism of glucose for their supply ofmetabolic energy. The mitochondrial citrate supplied can also betransported to cytoplasm by the tri or dicarboxcylic acid transporterwhere it can be converted to cytoplasmic acetyl CoA required for thesynthesis of acetyl choline. The reactions of the Krebs cycle are shownin Scheme 1 to help illustrate these concepts further.

[0042] Ketone bodies, in contrast to free fatty acids, cannot produceacetyl CoA in liver. Since acetyl CoA is the essential precursor offatty acid, they cannot result in either increased fatty acid orcholesterol synthesis in liver, which usually accounts for over half ofthe body's synthesis of these two potentially pathogenic materials.Liver is sensitive to the ratio of acetoacetate/(R)-3-hydroxybutyratepresented to it and will alter its mitochondrial free [NAD⁺]/[NADH],because of the near equilibrium established by β-hydroxybutyratedehydrogenase (EC 1.1.1.30) (31).

[0043] Inter alia, the aforementioned also indicates that one canprovide a method of increasing the efficiency of mitochondrial energyproduction in a human or animal not suffering from a chronic or acutemetabolic disease comprising administering to the human or animal anamount of a cyclic oligomer of formula (I) sufficient to raise bloodlevels of (R)-3-hydroxybutyrate to from 0.5 to 20 mM.

[0044] The easiest way to increase blood ketones is starvation. Onprolonged fasting blood ketones reach levels of 7.5 mM (62, 63).However, this option is not available on a long term basis, since deathroutinely occurs after a 60 day fast.

[0045] The ketogenic diet, comprised mainly of lipid, has been usedsince 1921 for the treatment of epilepsy in children, particularlymyoclonic and akinetic seizures (109) and has proven effective in casesrefractory to usual pharmacological means (71). Either oral orparenteral administration of free fatty acids or triglycerides canincrease blood ketones, provided carbohydrate and insulin are low toprevent re-esterification in adipose tissue. Rats fed diets comprised of70% corn oil, 20% casein hydrolysate, 5% cellulose, 5% McCollum's saltmixture, develop blood ketones of about 2 mM. Substitution of lard forcorn oil raises blood ketones to almost 5 mM (Veech, unpublished).

[0046] In general the levels of ketone bodies achieved on such diets areabout 2 mM (R)-3-hydroxybutyrate and 1 mM acetoacetate while the levelsof free fatty acids are about 1 mM. Other variations of composition havebeen tried including medium chain length triglycerides. In general,compliance with such restricted diets has been poor because of theirunpalatability (56). High lipid, low carbohydrate diets also have beentried as therapeutic agents in cancer patients to reduce glucoseavailability to tumors (88), as weight reducing diets in patients withand without diabetes (74, 112) and to improve exercise tolerance (83).

[0047] The limitation of diets which rely upon lipid to raise bloodketones to neurologically effective levels are many. Firstly, levels ofketone bodies on lipid based diets tend to be below 3 mM, significantlylower than the level of 7.5 mM achieved in overweight humans duringprolonged fasting. Secondly, unauthorized ingestion of carbohydrateincreases insulin secretion and causes a rapid decrease in the hepaticconversion of free fatty acids to ketones with a consequent drop inblood ketones and the diversion of lipid to esterified to triglyceridesby adipose tissue. Many anecdotal reports relate the resumption ofseizures in children who “broke their diet with birthday cake”. Thirdlytheir unpalatability and the necessity to avoid carbohydrate to sustainhigh ketone body levels makes such high lipid diets difficult to use inadults in an out patient setting, particularly in societies wheretraditionally high intake of refined sugars, bread, pasta, rice andpotatoes occurs. In practice, the traditional high ketone diet cannot beenforced in patients, other than children beyond the age where all foodis prepared at home under strict supervision. Fourthly, ingestion ofsuch large amounts of lipid in the adult population would lead tosignificant hypertriglyceridemia and hypercholesterolemia withpathological sequelae of increased vascular disease and sporadic hepaticand pancreatic disease, and therefore could not be prescribed on medicalgrounds. Ingestion of high lipid, low carbohydrate diets were popular inthe 1970s for weight reduction in the face of high caloric intake,provided that carbohydrate intake was low. However, because of theincreased awareness of the relationship of elevated blood lipids toatherosclerosis the popularity of this diet dropped abruptly.

[0048] Substituting glucose in a liquid diet, where glucose accounts for47% of the caloric content, with racemic 1,3 butandiol caused the bloodketone concentration to rise about 10 fold to 0.98 mM(R)-3-hydroxybutyrate and 0.33 mM acetoacetate (107). These values areslightly less than obtained normally in a 48 hour fast and far below thelevels of 7.5 mM obtained in fasting man. Racemic 1,3 butandiol isconverted by liver to acetoacetate and both the unnatural L-β and thenatural D-β-hydroxybutyrate (respectively (S) 3-hydroxybutanoate and(R)-3-hydroxybutyrate). Although racemic 1,3 butandiol has beenextensively studied as a cheap caloric source in animal food and haseven been used experimentally in human diets (81, 101) the production ofthe unnatural L-isomer is likely in the long run to produce significanttoxicity as has been shown for the human use of the unnatural D-lactate(64). One disadvantage of administering the unnatural L isomer is thatit competes for transport with the natural (R)-3-hydroxybutyrate. Thusprovision of the (R) 1,3 butandiol as a precursor of ketone bodies isone possibility that avoids unnecessary administration or production ofthe unnatural isomer.

[0049] The mono and di-aceotacetyl esters of racemic 1,3 butandiol havebeen suggested as a source of calories and tested in pigs (67). Oraladministration of a bolus of a diet containing 30% of calories as theesters produced a brief peak of blood ketones to 5 mM. However, the useof racemic 1,3 butandiol with its production of the abnormal (S)3-hydroxybutanoate is not to be recommended for the reasons statedabove.

[0050] While use of racemic 1,3 butandiol in such formulations is notrecommended, the esters of (R) 1,3 butandiol can be used, either aloneor as the acetoacetate ester. Studies in rats have shown that feedingracemic 1,3 butandiol caused liver cytosolic [NAD⁺]/[NADH] to decreasefrom 1500 to about 1000 (87). By comparison, administration of ethanolreduces hepatic [NAD−]/[NADH] to around 200 (106).

[0051] Acetoacetate, when freshly prepared, can be used in infusionsolutions where it can be given in physiologically normal ratios with(R)-3-hydroxybutyrate to optimum effect (95). Because of manufacturingrequirements which currently require long shelf life and heat sterilizedfluids, acetoacetate has frequently been given in the form of an ester.This has been done to increase its shelf life and increase its stabilityto heat during sterilization. In the blood stream, esterase activity hasbeen estimated to be about 0.1 mmol/min/ml and in liver about 15mmol/min/g (68). In addition to esters combining 1,3 butandiol andacetoacetate there has also been extensive study of glycerol esters ofacetoacetate in parenteral (59) and enteral nutrition (82). Suchpreparations were reported to decrease gut atrophy, due to the highuptake of acetoacetate by gut cells and to be useful in treatment ofbums (85).

[0052] For preferred embodiments of the present invention, under optimumconditions, a physiological ratio of ketones should be produced throughadministration of cyclic oligomers and acetoacetate. If it is not, inthe whole animal, the liver will adjust the ratio of ketones inaccordance with its own mitochondrial free [NAD⁺]/[NADH]. If an abnormalratio of ketones is given the liver will adjust the ratio, withcoincident changes in liver [NAD⁺]/[NADH]. In the working heart,perfusion with acetoacetate as sole substrate, rapidly induces heartfailure (99) in contrast to rat hearts perfused with a mixture ofglucose, acetoacetate and (R)-3-hydroxybutyrate, where cardiacefficiency was increased by a physiological ratio of ketone bodies (95).

[0053] The cyclic oligomers for use in the present invention areconveniently synthesized from the microorganism produced polyesters.Natural polyesters of (R)-3-hydroxybutyrate are sold as articles ofcommerce e.g. as polymers of 530,000 MW from Alcaligenes eutrophus(Sigma Chemical Co. St. Louis) or as 250,000 MW polymers for sugar beets(Fluka, Switzerland). The bacteria produce the polymer as a source ofstored nutrient. The fermentation of these polymers by bacteria wasdeveloped in the 1970s by ICI in the UK and Solvay et Cie in Belgium, asa potentially biodegradable plastic for tampon covers and other uses.The system responsible for the synthesis of the poly(R)-3-hydroxybutyrate has now been cloned and variations in thecomposition of the polymer produced, based on the substrates given tothe bacteria. The genes responsible for the synthesis of polyalkanoateshave been cloned and expressed in a number of micro-organisms (93, 102,113) allowing for production of this material in a variety of organismsunder extremely variable conditions.

[0054] Preferred forms of cyclic oligomeric (R)-3-hydroxybutyrate are,at least in part, readily digestable and/or metabolised by humans oranimals. These preferably are of 2 to 200 repeats, typically 2 to 20 andmost conveniently from 3 to 10 repeats long, particularly of 3 repeats,i.e. the triolide. It will be realised that mixtures of such oligomersmay be employed with advantage that a range of uptake characteristicsmight be obtained. Similarly mixtures with the monomer or linearoligomers or polymers may be provided in order to modify the blood levelprofile produced.

[0055] Cyclic oligomers for use in the invention may be provided, interalia, by methods described by Seebach et al. Helvetia Chimica Acta Vol71 (1988) pages 155-167, and Seebach et al. Helvetia Chimica Acta, Vol77 (1994) pages 2007 to 2033. For some circumstances such cyclicoligomers of 5 to 7 or more (R)-3-hydroxybutyrate units may be preferredas they may be more easily broken down in vivo. The methods of synthesisof the compounds described therein are incorporated herein by reference.

[0056] Once the monomer is in the blood stream, and since liver isincapable of metabolizing ketone bodies but can only alter the ratio of(R)-3-hydroxybutyrate/acetoacetate, the ketone bodies are transported toextrahepatic tissues where they can be utilized. The blood levels ofketones achieved are not subject to variation caused by noncompliantingestion of carbohydrate, as is the case with the present ketogenicdiet. Rather, they would simply be an additive to the normal diet, givenin sufficient amounts to produce a sustained blood level, typically ofbetween 0.3 to 20 mM, more preferably 2 to 7.5 mM, over a 24 hourperiod, depending upon the condition being treated. In the case ofresistant childhood epilepsy, blood levels of 2 mM are currently thoughtto be sufficient. In the case of Alzheimer's disease, attempts couldeven be made to keep levels at 7.5 mM or more, as achieved in thefasting man studies, in an effort to provide alternative energy andacetyl CoA supplies to brain tissue in Alzheimer's patients where PDHcapacity is impaired because of excess amounts of Aβ₁₋₄₂ amyloid peptide(77, 78).

[0057] The determination by the inventor that (R)-3-hydroxybutyrate andits mixtures with acetoactetate act as a nerve stimulant, e.g. nervegrowth stimulant and/or stimulant of axon and dendritic growth, opens upthe option of raising ketone body levels to lesser degrees than requirednutritionally in order to treat neurodegeneration.

[0058] Compositions of the invention are preferably sterile and pyrogenfree, particularly endotoxin free. Secondly, they are preferablyformulated in such a way that they can be palatable when given as anadditive to a normal diet to improve compliance of the patients intaking the supplements. The cyclic oligomers are generally smell free.Formulations of the cyclic oligomers of (R)-3-hydroxybutyrate and itsmixtures with acetoacetate may be coated with masking agents or may betargeted at the intestine by enterically coating them or otherwiseencapsulating them as is well understood in the pharmaceuticals ornutraceuticals art.

[0059] Since ketone bodies contain from about 4 to 6 calories/g, thereis preferably a compensatory decrease in the amounts of the othernutrients taken to avoid obesity.

[0060] Particular advantages of using the cyclic oligomers taught in thepresent invention are:

[0061] 1) they can be eaten with a normal dietary load of carbohydratewithout decreasing blood ketone body levels which decrease would impairthe effects of the treatment,

[0062] 2) they will not raise blood VLDL and cholesterol, as withcurrent cream and margarine containing diets, thus eliminating the riskof accelerated vascular disease, fatty liver and pancreatitis,

[0063] 3) they will have a wider range of use in a greater variety ofpatients, including but not limited to: type II diabetes to preventhypoglycemic seizures and coma, in Alzheimer's disease and otherneurodegenerative states to prevent death of nerve cells e.g.hippocampal cells, and in refractory epilepsy due to either decreases incerebral glucose transporters, defects in glycolysis, or so calledLeigh's syndromes with congenital defects in PDH.

[0064] The cyclic oligomers of the invention can be used in oral andparenteral use in emulsions, whereas acetoacetate, in the unesterifiedstate, is less preferred as it is subject to spontaneous decarboxylationto acetone with a half time at room temperature of about 30 days. Wherethe compositions of the invention do include acetoacetate this may be inthe form of a precursor. Acetoacetate may conveniently be provided as(R)-3-hydroxybutyrate esters as provided by the copending ‘therapeuticcompositions’ application.

[0065] Treatment may comprise provision of a significant portion of thecaloric intake of patients with the cyclic (R)-3-hydroxybutyrateoligomer or oligomers formulated to give retarded release, so as tomaintain blood ketones in the elevated range, e.g. 0.5 to 20 mM,preferably 2-7.5 mM range, over a 24 hour period. Release of the ketonebodies into the blood may be restricted by application of a variety oftechniques such as microencapsulation, adsorption and the like which iscurrently practised in the oral administration of a number ofpharmaceutical agents. Enterically coated forms targeting delivery poststomach may be particularly used where the material does not require, oris not susceptible to, hydrolysis in acid environment. Where some suchhydrolysis is desired uncoated forms may be used. Some forms may includeenzymes capable of cleaving the esters to release the ketone bodies suchas those referred to in Doi. Microbial Polyesters

[0066] Preferred cyclic oligomers, e.g. the triolide, may be merelyadded as such to foodstuffs and/or may be supplemented in a treatmentregime by other ketone body generators of different release profile suchas the monomeric (R)-3-hydroxybutyrate. The latter can be provided as anaqueous solution, e.g. as a salt, e.g. sodium, potassium, magnesium orcalcium salt

[0067] For a 1500 calorie diet, the human adult patient could consume198 g of cyclic esters of the present invention per day. For a 2000calorie diet of the same proportions, one could consume 264 g of ketonesper day. On the ketogenic lipid diet blood ketones are elevated to about2 mM, which proves to be effective to some degree at least in over 60%of children treated. On the ketone diet, ketone levels should be higherbecause ketones have been substituted at the caloric equivalent of fat,that is 1.5 g of ketone/ g of fat. Accordingly, blood ketones should beapproximately 3 mM, an effective level in children, but still below thelevel achieved in fasting man of 7.5 mM.

[0068] The advantage of using compounds which directly raised ketonebody levels, including the present cyclic oligomers which raise bloodlevels of ketone bodies themselves are several. Firstly, provision ofketone bodies themselves does not require the limitation ofcarbohydrate, thus increasing the palatability of the dietaryformulations, particularly in cultures where high carbohydrate diets arecommon. Secondly, ketone bodies can be metabolised by muscle, heart andbrain tissue, but not liver. Hence the fatty liver, which may be anuntoward side effect of the ketogenic diet, is avoided. Thirdly, theability to include carbohydrate in the dietary formulations increasesthe chance of compliance and opens up practical therapeutic approachesto type II diabetics where insulin is high, making the known ketogenicdiet unworkable.

[0069] The present inventor has determined that, while any elevation ofketone bodies may be desirable, a preferred amount of cyclic ester to beadministered will be sufficient, with any acetoacetyl component, toelevate blood ketone body levels to the 0.5 to 20 mM level, preferablyto the 2 mM to 7.5 mM level and above, particularly when attempting toarrest the death of brain cells in diseases such as Alzheimer's andParkinsonism. While dead cells cannot be restored, arrest of furtherdeterioration and at least some restoration of function is to beanticipated.

[0070] The total amount of ketone bodies used in treatment ofneurodegeneration such as Alzheimer's and Parkinsonism will preferablyelevate blood levels of ketone bodies by from 0.5 mM to 20 mM. Thepresent inventor estimates that 200 to 300 g (0.5 pounds) of ketonebodies equivalent per patient per day might be required to achieve this.Where the treatment is through maintenance of cells against the effectsof neurotoxin this may be at a level sufficient to act as a significantcaloric source, e.g. 2 to 7.5 mM in blood. Where it relies on the nervestimulatory factor effect of the (R)-3-hydroxybutyrate so produced, theamount administered may be lower, e.g. to provide 0.2 to 4 mM increase,but can of course be more for this or other disease.

[0071] It will be realised that treatment for neurodegenerative diseasessuch as Alzheimer's or Parkinsonism will most effectively be given soonafter identifying patient's with a predisposition to develop thedisease. Thus treatment for Alzheimers' most effectively follows apositive test result for one or more conditions selected from the group(i) mutations in the amyloid precursor protein gene on chromosome 21,(ii) mutations in the presenilin gene on chromosome 14, (iii) presenceof isoforms of apolipoprotein E. Other tests shown to be indicative ofAlzheimer's will of course be applicable.

[0072] Following such a positive test result it will be appropriate toprevent the development of memory loss and/or other neurologicaldysfunction by elevation of the total sum of the concentrations of theketone bodies (R)-3-hydroxybutyrate and/or acetoacetate in the patient'sblood or plasma to say between 1.5 and 10 mM, more preferably 2 to 8 mM,by one of several means. Preferably the patient is fed a diet ofsufficient quantities of compound of formula (I), optionallyparenterally but preferably and advantageously enterally.

[0073] It will be realised that hypoglycemic brain dysfunction will alsobe treatable using the treatments and compositions and compounds of thepresent invention. A further property associated with the presenttreatment will be general improvement in muscle performance.

[0074] The provision of cyclic oligomer based foodstuffs and medicamentsof the invention is faciliated by the ready availability of a number ofrelatively cheap, or potentially cheap, starting materials from whichcyclic (R)-3-hydroxybutyric acid may be derived (see MicrobialPolyesters Yoshiharu Doi. ISBN 0-89573-746-9 Chapters 1.1, 3.2 and 8).The availability of genes capable of insertion into foodstuff generatingorganisms provides a means for creating products such as yoghurts andcheese that are enriched in the cyclic oligomer-(R)-3-hydroxybutyricacid or, after breakdown with enzymes capable of cleaving such polymers,with the monomeric substance itself (see Doi. Chapter 8).

[0075] Methods of preparing poly (R)-3-hydroxybutyrate are notspecifically claimed as these are known in the art. For example Shang etal, (1994) Appli. Environ. Microbiol. 60: 1198-1205. This polymer isavailable commercially from Fluka Chemical Co. P1082, cat#81329,1993-94, 980. Second St. Ronkonkoma N.Y. 11779-7238, 800 358 5287.

[0076] The present invention will now be described further by way ofillustration only by reference to the following Figures and experimentalexamples. Further embodiments falling within the scope of the inventionwill occur to those skilled in the art in the light of these.

FIGURE

[0077]FIG. 1 is a graph showing blood (R)-3-hydroxybutyrate levelproduced after time after feeding rats with the triolide of(R)-3-hydroxybutyrate, a cyclic oligomer produced in Example 1 inyoghurt and controls fed yoghurt alone.

EXAMPLES Example 1 Preparation of(R,R,R)-4,8,12-trimethyl-1,5,9-trioxadodeca-2,6,10-trione: Triolide of(R)-3-hydroxybutyric Acid

[0078]

[0079] Synthesis was as described in Angew. Chem. Int. Ed. Engl. (1992),31, 434. A mixture of poly[(R)-3-hydroxybutyric acid] (50 g) andtoluene4-sulphonic acid monohydrate (21.5 g, 0.113 mole) in toluene (840ml) and 1,2-dichloroethane (210 ml) was stirred and heated to reflux for20 hours. The water was removed by Dean-stark trap for 15 hourswhereafter the brown solution was cooled to room temperature and washedfirst with a half saturated solution of sodium carbonate then withsaturated sodium chloride, dried over magnesium sulphate and evacuatedin vacuo. The brown semi-solid residue was distilled using a Kugelrohrapparatus to yield a white solid (18.1 g) at 120-130° C./0.15 mmHg.Above 130° C. a waxy solid began to distill— distillation being stoppedat this point. The distilled material had mp 100-102° C. (literature mp110-110.5° C.). Recrystallisation from hexane gave colourless crystalsin yield 15.3 g. Mp=107-108° C.; [α]_(D)-35.1 (c=1.005, CHCl₃),(lit.=−33.9). ¹H NMR (300 MHz, CDCl₃): δ=1.30 (d, 9H, CH₃); 2.4-2.6 (m,6H; CH₂); 5.31-5.39 (M, 3H; HC-O). ¹³C NMR (CDCl₃) δ=20.86 (CH₃), 42.21(CH₂), 68.92 (CH), 170.12 (CO). Elemental analysis: calculated forC₁₂H₁₈O₆: C, 55.81; H 7.02; Found: C, 55.67; H, 7.15.

Comparative Example 1 Preparation of Oligomers of (R)-3-hydroxybutyricAcid (R)-3-hydroxybutyrate)

[0080] (R)-3-hydroxybutyric acid (Fluka-5.0 g: 0.048 mole), p-toluenesulphonic acid (0.025 g) and benzene (100 ml) were stirred under refluxwithin a Dean-Stark trap arrangement for 24 hours. The reaction mixturewas cooled and the benzene evaporated in vacuo (0.5 mm Hg). 4.4 g ofcolourless oil was obtained of which a 20 mg sample was converted to themethyl ester for analysis of number of monomer repeats using NMR. Thesestudies show that the product is a mixture of oligomers ofD-β-hydroxybutyrate of average number of repeats 3.75, being mainly amixture of trimers, tetramers and pentamers with the single mostabundant material being the tetramer. The product mixture was soluble in1 equivalent of sodium hydroxide.

Comparative Example 2 Preparation of Acetoacetyl Ester of Oligomeric(R)-3-hydroxybutyric Acid

[0081] A further batch of the colourless oil product from Example 1 (4.5g) was heated for 1 hour at 60° C. with diketene (3.8 g) and sodiumactetate (0.045 g) under nitrogen. Further diketene (3.8 g) was addedand the reaction heated for a further hour, cooled and diluted withether, washed with water and then extracted with saturated sodiumbicarbonate (5×100 ml). Combined extract was washed with ether thenacidified with concentrated HCl (added dropwise). Ethyl acetateextraction (3×50 ml) was followed by drying over magnesium sulphate andevaporation in vacua. A yellow solid/oil mixture was obtained (7.6 g)which was chromatographed on a silica column usingdichloromethane/methanol (98:2) to give a light amber oil product.Faster moving impurities were isolated (1.6 g) and after recolumningcarbontetrachloride/methanol (99:1) 0.8 g of oil was recovered which wasshown by NMR and Mass spectrometry to be the desired mixture ofacetoacetylated oligomers of (R)-3-hydroxybutyrate. The product mixturehad an Rf of 0.44 in dichloromethane/methanol (90:1) and was soluble in1 equivalent of sodium hydroxide. Both products of Comparative Examples1 and 2 are amenable to separation of individual components bypreparative HPLC.

EXAMPLE 2 Oral Administration of Triolide of (R)-3-hydroxybutyrate ofExample 1 to Rats

[0082] The ability of orally administered triolide to raise blood ketonelevels was investigated as follows. The day before the experimentcommenced, 12 Wistar rats weighing 316±10 g were placed in separatecages. They had no access to food for 15 hours prior to presentationwith triolide containing compositions, but water was provided adlibitum.

[0083] On the morning of the experiment 0.64 g of triolide was mixedwith 5 g Co-op brand Black Cherry yoghurt in separate feeding bowls for9 of the rats. The remaining 3 rats were given 5 g of the yoghurtwithout the triolide as controls. The yoghurt containing bowls wereplaced in the cages and the rats timed while they ate. Two of the threecontrol rats ate all the yoghurt and four of the six triolide yoghurtrats ate approximately half the provided amount. The remaining six ratsslept.

[0084] Control rats (n=2) were killed at 60 and 180 minutes afteringestion of yoghurt while triolide fed rats were killed at 80, 140, 150and 155 minutes. Blood samples were taken for assay of(R)-3-hydroxybutyrate. Brains were funnel frozen and later extracted inperchloric acid and extracts neutralised and assayed. Blood levels of(R)-3-hydroxybutyrate were measured using a NAD⁺/EDTA assay of Anal.Biochem (1983) 131, p 478-482. 1.0 ml of a solution made up from2-amino-2-methyl-1-propanol (100 mM pH 9.9, 0.094 g/10 ml), NAD⁺ (30 mM,0.199 g/10 ml) and EDTA (4 mM, 0.015 g/10 ml) was added to each of anumber of cuvettes and 4 μl sample or (R)-3-hydroxybutyrate control.

[0085] The two control rats ate 5.2±0.1 g yoghurt and their plasma(R)-3-hydroxybutyrate concentrations were about 0.45 mM at 60 minutesand 180 minutes. The four triolide fed rats ate 0.39±0.03 g of thetriolide and 2.6±0.2 g of yoghurt. Their plasma (R)-3-hydroxybutyrateconcentrations were 0.8 mM after 80 minutes and 1.1 mM for the groupsacrificed at about 150 minutes. All rats displayed no ill effects fromingestion of triolide. Thus serum (R)-3-hydroxybutyrate was found to beelevated by 0.65 mM by feeding of only 0.4 g triolide. Note, as the ratshad been fasted, the initial levels of (R)-3-hydroxybutyrate wereelevated from the 0.1 mM fed state to about 0.45 mM.

[0086] The test rats thus showed increase in plasma(R)-3-hydroxybutyrate over at least 3 hours with no ill effects. Itshould be noted that two other rats fed approximately 1.5 g triolideeach in ‘Hob-Nob’ biscuit showed no ill effects after two weeks.

[0087] It should be noted that the increased levels of(R)-3-hydroxybutyrate will also be mirrored in acetoacetate levels, notmeasured here, as there is a rapid establishment of equilibrium betweenthe two in vivo such that acetoacetate levels will be between 40 and100% of the (R)-3-hydroxybutyrate levels.

Comparative Example 3 Oral Administration of (R)-3-hydroxybutyrateOligomers and Acetoacetyl (R)-3-hydroxybutyrate Oligomers to Rats

[0088] The ability of orally administered (R)-3-hydroxybutyrate and thelinear oligomers of Comparative examples 1 and 2 to raise blood ketonebody levels was investigated as follows. Rats were fasted overnight andthen gavaged with 100 μl/100 g bodyweight of 4M (R)-3-hydroxybutyratebrought to pH 7.4 using methyl glucamine. Plasma levels of(R)-3-hydroxybutyrate were measured at 0.62 mM after 30 minutes ascompared to 3 mM when 9M (R)-3-hydroxybutyrate is used.

[0089] This procedure was repeated with 2M solutions of the mixtures(R)-3-hydroxybutyrate oligomers and their acetoacetyl esters describedin Comparative Examples 1 and 2. The (R)-3-hydroxybutyrate oligomer(19/1) and the acetoacetyl ester (20/4) were both brought to pH 7.6 withmethyl glucamine and the blood (R)-3-hydroxybutyrate level monitoredusing the aforesaid assay procedure. Increases in serum(R)-3-hydroxybutyrate were shown to be of 0.2 mM to 0.5 mM at 60 and 120minutes after gavaging.

EXAMPLE 5

[0090] TABLE 2 Sample 1500 calorie ketogenic diet using cyclic oligomer(I) of invention. The cyclic oligomer is assumed to contain 6 kcal/gfats, 9 kcal/g carbohydrate and 4 kcal/g protein. Oligomers have beensubstituted to give equilvalent calories Protein Cyclic Amount (g) Fat(g) (g) CHO (g) (I) (g) Breakfast Egg 32 4 4 apple juice 70 7 ketones 6666 skim milk 92 0 2 3 Total Breakfast 4 6 10 66 Lunch lean beef 12 1.753.5 cooked carrots 45 0.6 3 canned pears 40 4 ketones 69.75 69.75 skimmilk 92 2 3 Total Lunch 1.75 6.1 10 69.75 Supper Frankfurter 22.5 6 3cooked broccoli 50 1 2 watermelon 75 5 ketones 62.25 62.25 skim milk 922 3 Total Supper 6 6 10 62.25 Daily Total 11.75 18.1 30 198

EXAMPLE 6 Effect of (R)-3-hydroxybutyrate on Hippocampal Cells Methods

[0091] Culture Medium and Chemicals

[0092] The serum free medium used from 0 to day 4 contained Neurobasalmedium with B27 supplement diluted 50 fold (Life Technology,Gaithersburg, Md.) to which was added: 0.5 mM L-glutamine, 25 μM NaL-glutamate, 100 U/ml penicillin and 100 μg/ml streptomycin. After day4, DMEM/F12 medium containing 5 μM insulin, 30 nM 1-thyroxine, 20 nMprogesterone, 30 nM Na selenite 100 U/ml penicillin and 100 μg/mlstreptomycin were used.

[0093] Hippocampal Microisland Cultures

[0094] The primary hippocampal cultures were removed from Wistar embryoson day 18 and dispersed by gentle agitation in a pipette. The suspensionwas centrifuged at 1,500×g for 10 min and the supernatant discarded. Thepellet was resuspended in new media to a final cell count of 0.4-0.5×10⁶cells/ml. Ten μl of this suspension was pipetted into the center of polyD-lysine coated culture wells and the plates incubated at 38° C. for 4hrs and then 400 μl of fresh Neurobasal media was added. After 2 days ofincubation, half of the media was exchanged for fresh media and theincubation continued for 2 more days. After day 4, the medium waschanged with DMEM/F12 medium containing 5 μM insulin, 30 nM 1-thyroxine,20 nM progesterone, 30 nM Na selenite 100 U/ml penicillin and 100 μg/mlstreptomycin. The wells were divided into 4 groups: half the wellsreceived (R)-3-hydroxybutyrate to a final concentration of 8 mM whileand half of the wells received 5 nM amyloid β₁₋₄₂ (Sigma). These mediawere exchanged 2 days later (day 8) and the cells were fixed on day 10and stained with anti MAP2 (Boehringer Manheim, Indianapolis, Ind.) tovisualize neurons and vimentin and GFAP (Boehringer) to visualize glialcells.

Results

[0095] Cell Counts

[0096] Addition of (R)-3-hydroxybutyrate to the incubation resulted inan increase in the neuronal cell number per microisland from a mean of30 to a mean of 70 cells per microisland. Addition of 5 nM amyloid β₁₋₄₂to the cultures reduced the cell numbers from 70 to 30 cells permicroisland, confirming the previous observations of Hoshi et al, thatamyloid β₁₋₄₂ is toxic to hippocampal neurons. Addition of(R)-3-hydroxybutyrate to cultures containing amyloid β₁₋₄₂ increased thecell number from a mean of 30 to 70 cells per microisland. From thesedata we conclude that addition of substrate level quantities of(R)-3-hydroxybutyrate, to media whose major nutrients are glucose,pyruvate and L-glutamine, slows the rate of cell death in culture. It isfurther concluded that (R)-3-hydroxybutyrate can decrease the increasedrate of hippocampal cell death caused by the addition of amyloid β₁₋₄₂in culture.

[0097] The number of dendritic outgrowths and the length of axons wereboth observed to have increased with presence of(R)-3-hydroxybutyrate,whether β₁₋₄₂ was present or not. This is indicative of nerve growthfactor like behaviour.

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1. A cyclic ester of (R)-3-hydroxybutyrate of formula (I)

where n is an integer of 1 or more or a complex thereof with one or morecations or a salt thereof for use in therapy or nutrition.
 2. A compoundas claimed in claim 1 wherein the one or more cations are selected fromthe group consisting of sodium, potassium, magnesium and calcium orwhere the compound is a free uncomplexed oligolide.
 3. A compound asclaimed in claim 1 or claim 2 wherein n is an integer from 1 to to 20.4. A compound as claimed in claim 1 wherein it is(R,R,R)-4,8,12-trimethyl-1,5,9-trioxadodeca-2,6,10-trione.
 5. A methodof treating a cell that is subject to malfunction due to action of freeradicals, toxic agents such as peptides and proteins and genetic defectsdeleterious to cell metabolism, insulin resistance or other glucosemetabolism defects or defect inducing states, ischemia, head trauma,and/or for increasing cell efficiency characterised in that it comprisesadministration of a cyclic oligomer of formula (I).
 6. A method asclaimed in claim 4 characterised in that the cyclic oligomer of formula(I) acts as a neuronal stimulant e.g. capable of stimulating axonaland/or dendritic growth in nerve cells, e.g. in hippocampal orsubstantia nigral cells, in vivo or in vitro, particularly in conditionswhere neuro-degeneration has serious clinical consequences.
 7. A methodof accomplishing enteral or parenteral nutrition, preferably oral routenutrition, comprising administration of a cyclic oligomer of formula (I)in physiologically acceptable form, optionally in a physiologicallyacceptable carrier.
 8. A method of producing a physiologicallyacceptable ketosis in a human or animal comprising oral administrationof a cyclic oligomer of formula (I).
 9. A method as claimed in claim 8wherein the human or animal is fed less than 50% by weight of itscaloric content of its diet as fat.
 10. A method as claimed in claim 8wherein the human or animal is fed from 0 to 25% by weight of itscaloric content of its diet as fat.
 11. A method as claimed in claim 7or 8 wherein the method is performed on a patient needing therapy forone or more of Alzheimer's, Parkinsonism, Amylotrophic lateralsclerosis, Epilepsy, Free radical disease, Heart failure, Type IIdiabetes, deficiency or blockage of pyruvate dehydrogenase, inability toperform glycolysis in one or more cell types and Duchenne's musculardystrophy.
 12. A method of providing a caloric substitute forcarbohydrate for the purpose of lowering blood glucose comprisingadministering a composition comprising a cyclic oligomer of formula (I)to a human or animal subject in need of such substitution.
 13. A methodof providing a caloric substitute for carbohydrate for the purpose ofbody lipid content reduction comprising administering a compositioncomprising a cyclic oligomer of formula (I) to a human or animal subjectin need of such reduction.
 14. A method of increasing the efficiency ofmitochondrial energy production in a human or animal not suffering froma chronic or acute metabolic disease comprising administering to thehuman or animal an amount of a cyclic oligomer of formula (I) sufficientto raise blood levels of (R)-3-hydroxybutyrate to from 0.5 to 20 mM. 15.A method as claimed in claim 14 wherein the level is raised to from 1 to10 mM.
 16. The use of a cyclic ester formula I for the manufacture of amedicament for the treatment of disease states mediated by freeradicals, toxic agents such as peptides and proteins, genetic defectsdeleterious to cell metabolism, insulin resistance or other glucosemetabolism defects or defect inducing states, ischemia, head trauma, orfor increasing cell efficiency.
 17. A composition characterised in thatit comprises a cyclic oligomer of formula (I) in physiologicallyacceptable form.
 18. A composition as claimed in claim 11 characterisedin that it includes a physiologically acceptable carrier.